Abstract: The aim of this study was to establish a method for detection of selamectin in dog plasma by using doramectin as an internal standard. Samples were measured by high-performance liquid chromatography with fluorescence detection after automated solid phase extraction. After proteins separated with acetonitrile, a solid phase extraction was performed to plasma samples. The eluate was evaporated to dryness under stream of nitrogen gas, and followed by chemical derivation using N-methylimidazole and trifluoroacetic anhydride. An aliquot sample (20 μL) was pumped into the chromatographic column connected with fluoresce detector that wavelength was set at 355 nm for excitation and 465 nm for emission at 30℃. The mobile phase composed of methanol-acetonitrile-sodium 1heptanesulfonate(1%)acetic acid(0.4%) (40.0:57.6:0.9:1.5, v/v/v/v) were used, with flow rate 1 mL·min-1. The method gave linear calibration graphs (R2=0.999 3) over the concentration range studied (0.550.0 ng·mL-1) with detection limit, variation coefficient and average recovery of samples were 0.25 ng·mL-1, 1.47%3.31% and 94.00% respectively. It indicated that the method we established was reliable, sensitive and with good repeatability and suitable for detection of selamectin in dog plasma.